This invention relates to a chemical modification of biologically active proteins which alters the chemical and/or physiological properties of these proteins. More specifically, this invention relates to selective conjugation of a poorly water-soluble protein to a modified polyproline homopolymer to render the protein more soluble at physiological pH.
Many heterologous proteins produced in microbial host cells are found as poorly soluble material in refractile bodies. Examples of heterologous proteins which form refractile bodies in commonly found culture conditions include interleukin-2 (IL-2), interferon-.beta. (IFN-.beta.), feline leukemia virus (FeLV) envelope protein, human growth hormone (hGH), bovine growth hormone (bGH), porcine growth hormone (pGH), and certain proteins coated or fused with a virus such as FMD virus. In addition, many of these proteins are hydrophobic in nature and tend to stick to materials and to themselves (i.e., aggregate) rather than remain in solution. Also, many of these recombinant proteins are unglycosylated, whereas their native counterparts are water-soluble, glycosylated molecules. Modifications of these proteins that might alter their solubility properties would be desirable to increase production yields of these proteins as well as to facilitate their forrulation for therapeutic use.
The use of polypeptides in circulatory systems for the purpose of producing a particular physiological response is well known in the medicinal arts. A limitation to the potential therapeutic benefit derived from the clinical use of polypeptides is their ability to elicit an immune response in the circulatory system. This immune 30 response may be caused by aggregates in the material prior to injection as described by R. Illig (1970), J. Clin. Endrocr., 31, 679-688, W. Moore (1978), J. Clin. Endrocrinol. Metab., 46, 20-27 and W. Moore and P. Leppert (1980), J. Clin. Endrocrinol. Metab., 51, 691-697. This response involves the production of antibodies to the polypeptides by the circulatory system into which they are injected. This antibody production may decrease or eliminate the desired biological function of the polypeptide, sometimes by causing reduced residence time in the circulatory system (reduced half-life) or by modifying the molecule by virtue of the antibody-polypeptide interaction.
Modification of these potentially useful therapeutic polypeptides, per se, may increase the half-life of the polypeptides. The mechanism for this increase is not clear; it does not involve the immune system, but it may be due to clearance or protein turnover mechanisms.
The problems of immunogenicity and short half-life in circulation set forth hereinabove and other undesirable properties of certain proteins are well recognized and various modifications of polypeptides have been undertaken to solve them. These include the modification of proteins with substantially straight chain polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG).
For example, U.S. Pat. No. 4,055,635 discloses pharmaceutical compositions comprising a water-soluble complex of a proteolytic enzyme linked covalently to a polymeric substance such as polysaccharides.
U.S. Pat. No. 4,088,538 discloses a reversibly soluble enzymatically active polymer enzyme product comprising an enzyme covalently bonded to an organic polymer such as polyethylene glycol.
U.S. Pat. No. 4,415,665 discloses a method of conjugating an organic ligand containing at least one primary or secondary amino group, at least one thiol group and/or at least one aromatic hydroxy group (described in col. 3, lines 19-36) to a polymeric carrier with at least one hydroxyl group (described in col. 2, lines 42-66).
U.S. Pat. No. 4,496,689 discloses a covalently attached complex of alpha-1-proteinase inhibitor with a polymer such as PEG or methoxypolyethylene glycols.
U.S. Pat. No. 3,619,371 discloses a polymeric matrix having a biologically active substance chemically bound thereto.
U.S. Pat. No. 3,788,948 discloses use of organic cyanate pounds to bind proteins to polymers.
U.S. Pat. No. 4,055,635 discloses pharmaceutical compositions of a proteolytic enzyme linked covalently to a polymeric substance.
JP 5792435 published November 26, 1982 discloses modified polypeptides where all or part of the amino groups are substituted with a polyethoxyl moiety. DE 2312615 published September 27, 1973 discloses conjugating of polymers to compounds containing hydroxy or amino groups.
EP 147,761 discloses a covalent conjugate of alpha-1-proteinase inhibitor and water-soluble polymer, where the polymer may be polyethylene glycol.
EP 154,316, published September 11, 1985 to Takeda Chemical Industries, Ltd., discloses and claims chemically modified lymphokines such as IL-2 containing PEG bonded directly to at least one primary amino group of a lymphokine.
U.S. Pat. No. 4,414,147 describes rendering-interferon less hydrophobic by conjugating it to an anhydride of a dicarboxylic acid such as poly(ethylene succinic anhydride).
Copending U.S. Ser. No. 866,459 filed May 21, 1986 discloses-conjugation of PEG and polyoxyethylated polyols to such proteins as interferon-.beta., interleukin-2 and immunotoxins.
Copending U.S. Ser. No. 879,456 filed June 27, 1986 discloses conjugation of heparin fragments to such proteins as interforon-.beta., interleukin-2, and immunotoxins.
Chemotherapeutic agents such as melphalan have been conjugated through a carbodiinide-catalyzed reaction to poly(amino-acids) to yield drug carriers that may attain sustained release in cancer chemotherapy. See Morimoto et al., J. Pharm. Dyn., 7, 688-698 (1984).
Copending U.S. Ser. No. 648,759 filed Sept. 7, 1984, discloses a toxin conjugate for preparing immunotoxins wherein the cloned bridge between the toxin portion and the antibody portion is a sequence of 4-6 proline residues bracketed by two flexible amino acid sequences. The bridge is designed to be non-hydrophobic to maintain or improve water solubility of the immunotoxin.
The Morimoto et al. article and the other references do not disclose how to conjugate polyproline to recombinant proteins such as IL-2 and IFN-.beta. that are not very soluble in an aqueous medium at physiological pH. Furthermore, it is not a priori possible to predict which selected proteins would be favorably responsive, e.g., have good biological activity, to treatment with polyproline, due to the vast difference in physical and biological properties among proteins.